Advantages of transgenic animals before microorganisms-producers of biological products
Transgenic animals used for the production of valuable biologics have several advantages over microorganisms-producers, as well as cellular systems. In simple recombinant systems of microorganisms, the glycosylation, B-hydroxylation or carboxylation of mammalian proteins is in most cases impossible or possible, but with insufficient accuracy. This leads to a change in the structure of proteins, which can not but affect their biological activity. Along with this, in preparations used by man as a therapeutic agent, an admixture of bacterial proteins is undesirable. The main disadvantage of genetically engineered culture cells is the low yield of protein. Industrial reactors used to cultivate producer cells are expensive, both in terms of their cost and in terms of their maintenance. The creation of transgenic animals also requires a lot of money and besides it's not an easy thing, but once deduced a line of such animals can produce a large number of proteins with low cost, which will pay back all costs in a short time. Production of biologically active human proteins from transgenic agricultural products.animals guarantees their ecological purity and ecological purity of the production itself, which practically reduces to the exploitation of animal producers.
How do you know the recombinant proteins obtained from the milk of farm animals?
Alien proteins can be synthesized by most tissues of the animal's body. Expression of transgenes in certain organs can be achieved by combining structural genes with specific regulatory elements. Significant progress in the production of bioreactors has been achieved in the targeted expression of transgenes in mammary epithelial cells. The structural gene associated with the promoter of the milk protein gene (casein, lactalbumin, lactoglobulin) will primarily be expressed in mammary cells. This allows you to get useful products with milk.
The choice of a breast as a place of production of foreign proteins is justified by its enormous protein productivity. The total content of milk proteins, depending on the type of animal, varies between 2-10%, i.e. at a level of 20-100 grams per liter. For the commercial production of proteins that are of pharmaceutical importance, one or more grams of recombinant protein is sufficient. The most effective "bioreactor" is cattle, which annually at a yield of 5 thousand liters. milk, can give approximately 35 g protein per 1 liter. If the milk contains such a quantity of recombinant protein and the efficiency of its purification is 50%, then from 20 transgenic cows it will be possible to receive 50 kg of such protein per year. Figuratively speaking, two cows are sufficient to fully meet the annual requirement for protein C, used to prevent thrombus formation, and factor IX - (factor Christmas) cascade mechanism of blood coagulation.
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To date, a number of recombinant proteins are known, such as human protein C, anti-hemophilic factor 1X, alpha-1-antitrypsin, tissue plasma activator, lactoferin, human serum albumin, interleukin-2, urokinase, chymosin, etc., derived from the milk of transgenic animals . The work to produce these proteins, with the exception of alpha-1-antitrypsin and chymosin, is at the level of laboratory studies and has not reached a stage that would be of commercial interest. One of the goals of transgenosis of cattle is the change in the content of milk in various components. Thus, the amount of cheese obtained from milk is directly proportional to the content of k-casein in it, so it seems very promising to increase the amount of synthesized k-casein by overexpression of the transgene of this protein.
In 1992, scientists from the United Kingdom obtained transgenic sheep - producers of human alpha-1-antitrypsin, used to treat people with emphysema. This drug is obtained exclusively from donor blood (1 g of alpha-1-antitrypsin costs 110 US dollars). In four heads, the concentration of this protein was within 1 g / l, and one reached 35 g / l, which corresponds to half of all proteins in milk. At this level of production, one sheep per year will yield as much protein as is needed to treat 50 patients.
Russian scientists (LK Ernst, G. Brem, M. I. Prokofiev, I. L. Gol'dman, and others) received transgenic sheep secreting chymosin enzyme with milk at a concentration of 200-300 mg / l. Chimozin is the main component in the production of cheese, obtained from the rennet of dairy calves and lambs. At the same time the cost of chymosin, obtained from a new source, will be cheaper by 5-10 times. According to the calculations of the authors of three liters of milk transgenic sheep, you can get such an amount of enzyme, which is enough to produce one ton of cheese from cow's milk.
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Expression of transgenes in the cells of the mammary glands of sheep and goats did not have any side effects either on the females during the lactation period or on the feeding offspring. In contrast, when a bovine growth hormone was administered to pigs, under the control of the metallothionein promoter, adverse effects were observed. The amount of the hormone in the different individuals in the group of transgenic pigs was different, but in general, the whole group quickly gained weight. Unfortunately, this positive result was partially devalued by various pathologies: in animals, stomach ulcers, renal failure, lameness, pericardial inflammation, decreased mobility of joints, predisposition to pneumonia were noted. The causes of these symptoms are unknown. Perhaps, they are associated with the long-term presence in the body of excess growth hormone. In these experiments, the transgene was synthesized more or less continuously. Transgenic sheep were also created with increased growth rate of wool. To do this, the sheep insulin-like growth factor I cDNA was placed under the control of the mouse promoter of the keratin gene with a high sulfur content, which provided overexpression of the cDNA. In transgenic sheep, unlike pigs, no undesirable side effects were observed.
Positive results were also obtained during experiments with transgenic pigs. For example, healthy transgenic pigs were created in the genome of which the following genetic construct was present: the regulatory region of the human beta-globin gene, two human alpha-1 globulin genes and one human beta-globin gene. As a result of its expression in blood cells of pigs, human hemoglobin was synthesized, and as a result of replacing the human promoter of the beta-globin gene with pigs, human hemoglobin was synthesized in a much larger amount. Human hemoglobin, produced by transgenic pigs, had the same chemical properties as natural human. It could be purified from hemoglobin of pigs by usual chromatography.
These results indicate the principal possibility of replacing the whole blood used in transfusion with human hemoglobin obtained by the method of transgenesis. However, isolated hemoglobin transfers oxygen not as efficiently as hemoglobin in the composition of erythrocytes. Moreover, it quickly degrades in the body of the animal, which was introduced, and its decay products are toxic to the kidneys. Thus, obtaining a human blood substitute with transgenesis is a matter of the distant future.
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Recently, much attention has been paid to the use of animal organs for human transplantation. The main problem of interspecific transplantation is a hyperacute rejection. Hyperostroic rejection entails the binding of antibodies of the host organism to the carbohydrate antigenic determinant on the surface of the cells of the transplanted organ. The bound antibodies cause an acute inflammatory reaction (activation of the complement cascade), mass death of the antibody-carrying cells occurs and rapid loss of the transplanted organ.
In natural conditions, the inflammatory reaction is blocked by special proteins on the surface of cells lining the walls of blood vessels. These proteins - complement inhibitors are species-specific. It has been suggested that if the donor animal carried one or more genes of the human complement protein, the transplanted organ would be protected from the primary inflammatory response. To this end, transgenic pigs bearing various human complement inhibitor genes were obtained. The cells of one of these animals proved completely insensitive to the components of the complement cascade system. Preliminary experiments on the transplanting of organs of transgenic pigs to primates have shown that the tissues of the transplanted organ are weakened less, and the organ itself does not tear away a little longer. Perhaps the transgenic pigs carrying the human complement complement gene and lacking the basic surface protein of pig cells that causes severe rejection will serve as a source of organs for human transplantation.
Encouraging was the first work on obtaining transgenic animals - producers of interleukin-2. The latter, being a soluble factor of T-lymphocytes of helpers involved in the proliferation and differentiation of killer T-lymphocytes, plays an important role in providing the necessary level of immunity. Using a gene construct consisting of rabbit beta-casein DNA and human interleukin-2 structural gene, rabbits were obtained secreting the active form of this protein with milk.
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Thus, integration of one or several genes into mammalian embryos has been achieved and their expression has been proven, as well as transmission to offspring. However, it is necessary to emphasize the difficulties and ambiguities with which the technique of producing transgenic animals is still connected. The mechanism of gene integration into mammalian cells is still poorly understood. This integration occurs randomly and is not associated with a particular region of the chromosome. Another complication is caused by the instability of cells into which a gene (genes) is introduced: it can be lost or modified and, as a result, become inactive. Finally, the activity of genes is determined not only by nucleotide sequences that provide transcription of the gene with the formation of mRNA, but also by other nucleotide sequences that are often far away from their own gene, and if complete gene expression is required, these sequences need to be introduced along with the gene. For example, the gene responsible for the synthesis of alpha-globulin is regulated by the DNA sequence located in front of it.
The results of genetic engineering in the field of obtaining transgenic mammals will allow us to deepen our knowledge of gene expression, which in the future will facilitate the transfer of genes and the identification of factors that contribute to a more complete manifestation of genetic information recorded in transgenes. In addition, the insertion of a foreign gene into that region of the cell genome, where a homologous gene normally resides, will probably open the way for the treatment of genetic diseases, since this will allow replacing the defective gene or replacing the missing gene with its active analogue.
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